Sequence-specific isothermal and polymerase chain reaction (PCR) nucleic acid amplification techniques represent a rapidly growing sector of molecular diagnostics, offering rapid, sensitive detection of DNA samples.
Several isothermal techniques require multiple enzymes to work in concert, for example, strand displacement amplification (SDA), helicase dependent amplification (HDA), and isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN). Loop mediated isothermal amplification (LAMP) provides sequence-specific amplification using only a strand-displacing DNA polymerase (Gill and Ghaemi, Nucleosides, Nucleotides, and Nucleic Acids, 27:224-43 (2008); Kim and Easley, Bioanalysis, 3:227-39 (2011)). In addition to the DNA polymerase, LAMP uses 4 core primers (FIP, BIP, F3, and B3) recognizing 6 distinct sequence regions on the target (FIG. 1(a)-(b)), with two primers containing sequence (F1C, B1C) that results in loop structures which facilitate exponential amplification (Notomi, et al., Nucleic Acids Res., 28:E63 (2000)). The use of multiple target sequence regions confers a high degree of specificity to the reaction. Two additional primers, termed loop primers, can be added to increase reaction speed, resulting in 6 total primers used per target sequence (Nagamine, et al., Mol. Cel. Probes, 16:223-9 (2002)). The LAMP reaction rapidly generates amplification products as multimers of the target region in various sizes, and is substantial in total DNA synthesis (>10 μg, >50×PCR yield) (Notomi, et al. (2000); Nagamine, et al., Clin. Chem., 47:1742-3 (2001)) (see FIG. 1(a)-(b)).
Measurement of LAMP amplification product may be performed using fluorescence detection of double-stranded DNA (dsDNA) with an intercalating or magnesium-sensitive fluorophore (Notomi, et al. (2000); Goto, et al., Biotechniques, 46:167-72, (2009)), bioluminescence through pyrophosphate conversion (Gandelman, et al, PloS One, 5:e14155 (2010)), turbidity detection of precipitated magnesium pyrophosphate (Mori, et al., Biochem. Biophys. Methods 59:145-57 (2004); Mori, et al., Biochem. Biophys Res. Commun., 289:150-4 (2001)), or even visual examination through precipitated Mg2P2O7 or metal-sensitive dye (Tomita, Nat. Protoc., 3:877-82 (2008); Tao, et al., Parasit Vectors, 4:115 (2011)). These methods are robust and familiar, and visual methods are ideal for use in field diagnostics, but detect total DNA amplification in a reaction and are thus limited to detection of a single target. As isothermal techniques are further adopted as diagnostic tools, the ability to detect multiple targets in a single sample is desirable. Currently, quantitative, real-time PCR (qPCR) enables probe-specific multiplex detection and the ability to perform tests with an internal standard for definitive negative results. However, qPCR probes require extensive design and optimization for use and may not effectively translate to the LAMP reaction (Holland, et al., Proc Natl Acad Sci USA, 88:7276-80 (1991); VanGuilder, et al., Biotechniques 44:619-26 (2008); Didenko, Biotechniques 31:1106-16, 1118, 1120-1 (2001); Bustin, A-Z of Quantitative PCR. International University Line, La Jolla, Calif. (2006)).
Samples containing several different DNAs of interest have been analyzed using endpoint agarose gel electrophoresis (Aonuma, et al., Exp Parasitol, 125:179-83 (2010); He, et al., Aquaculture, 311:94-99 (2010)) or pyrosequencing (Liang, et al., Anal Chem, 84:3758-63 (2012)) but these do not allow real-time detection and require additional processing and instrumentation. In addition, the sensitivity of LAMP reactions to carryover contamination is so great that manufacturer recommendations (Eiken Chemical, Tokyo, Japan) suggest not opening LAMP reaction vessels, or doing so in separate facilities with separate equipment, further decreasing the desirability of post-LAMP manipulation. Previous real-time methods use non-specific quenching, either through loss-of-signal guanine quenching (Zerilli, et al., Clin Chem, 56:1287-96 (2010)) or gain-of-signal fluorescence using labeled primers and an intercalating dye (Kouguchi, et al., Mol Cell Probes, 24:190-5 (2010)). These methods can be less sensitive, and nonspecific quenching limits the selection of fluorophores available for multiplexing.
PCR requires a pair of primers and thermophilic DNA polymerase such as Taq DNA polymerase. During amplification, cycles of denaturation, annealing and primer extension steps allow primers to bind to the target sequence and DNA synthesis. Two types of detection are commonly used: endpoint or real time. A typical endpoint detection is agarose gel electrophoresis that allow identification of the specific target based on amplicon size and the yield. Realtime PCR or qPCR monitors the DNA production while the target DNA are being amplified.
The detection of qPCR can be divided into two types: the first type uses a double strand DNA intercalating dye and the second type uses a sequence specific probes. A number of methods have been described using sequence-specific probe (Holland, et al. (1991); VanGuilder, et al. (2008); Didenko (2001); Bustin (2006)). However, these typically require design of fluorescent probes in addition to the PCR primers.